I propose to study polypeptide-secreting and circadian oscillator neurons in tissue culture as dissociated cell systems from the opisthobranch mollusk Aplysia. The intact but isolated eye of Aplysia has a build-in neuronal circadian oscillator which can free-run for about two weeks in vitro, in darkness. Entrainment and temperature compensation of the Circadian period are inherent mechanisms within the eye. We will dissociate the eye into individual cells in order to localize the circadian oscillator. Light and electron-microscopic studies of such dissociated cells will be performed for identification purposes. Short-term intracellular and long-term extracellular electrical recordings will be used to identify neuron types involved in ultradian and circadian rhythms. Optical means of recording membrane activity through light absorption by membrane-bound dyes will be attempted. Time-lapse motion picture studies of nuclear and cytoplasmic changes in these neurons over the 24 hour period will be obtained. The bag cell neurons synthesize and release a 6000 molecular weight polypeptide which induces behavioral egg-laying. The special developmental, electrophysiological and biochemical properties of this peptidergic system will be studied in tissue culture where single neurons and small connected clusters can be examined under more rigorously controlled conditions.